Background/aims: Rotavirus-associated gastroenteritis is one of the main reasons for morbidity and mortality in Arab populations. The current investigation aimed to analyze distinctive structural mutations of VP4 and VP7 protein sequences from Saudi Arabia and the determination of the phylogenetic relationship using an evolutionary model. Materials and methods: Query and reference sequences were mined from a viral database of NCBI. Sequence variation analysis, phylogenetic analysis, intertaxon clade classification, the functional impact of mutation mutational stability analysis, and evolutionary conservation determination were executed by employing multiple sequence alignment version 3.8.31 (MUSCLE: v.3.8.31)/Clustal Omega (V2.1), iterative Tree of Life (iTOL: v5), Predict SNP & MutPred2, I-Mutant version 3.0/support vector machine (SVM)/MUpro, and ConSurf web-server respectively. Results: Among thirty-five mutational sites in total, with predicted conservation scores of two sites (I108T, S258A) of VP7 protein and five sites (G145N, I123V, Y102S, D110N, and T195K) of VP4 protein were identified as deleterious. Mutational sites (D110N and S258A) were found to decrease the stability of the VP4 and VP7 proteins. D110N and S258A were found to impact the protein function (by decreasing the stability) as variable mutation positions are subjected to variable protein functions. Conclusion: Understanding the pathogenesis relies on structural mutations in protein VP4 and VP7 sequences. Pathogenic potential and stability of 35 variations, and deleterious  D110N and S258A variations were ascertained. This could be used to design a vaccine construct for rotavirus serving as a potential treatment. These discoveries are important because the mutation may be able to confer co-protection against all rotavirus.